Paper : 03 Structure and Function of Biomolecules II

Module: 14 DNA Polymerase

Principal Investigator Dr. Sunil Kumar Khare, Professor,

Department of Chemistry, IIT-Delhi

Paper Coordinator and Dr. Sunil Kumar Khare, Professor,

Content Writer Department of Chemistry, IIT-Delhi

Content Reviewer Prof. Arun Goyal, Professor, Department

of Biochemical Engineering and
Biotechnology, IIT-Guwahati

Structure and Function of Biomolecules II

Biochemistry
DNA Polymerase
 DESCRIPTION OF MODULE

Subject Name Biochemistry

Paper Name 03 Structure and Function of Biomolecules II

Module Name/Title 14 DNA Polymerase

Dr. Vijaya Khader

Dr. MC Varadaraj

Structure and Function of Biomolecules II

Biochemistry
DNA Polymerase
 1. Objectives

 Function of DNA polymerase in DNA replication

 Mechanism of DNA polymerase catalysis
 Different types of Bacterial and Eukaryotic DNA polymerases
 Unique DNA polymerases

2. Concept Map

Structure and Function of Biomolecules II

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 3. Description

3.1 Overview

Deoxyribonucleic acid (DNA) is the genetic material in most of the organisms. To pass on the

genetic information during cell division, DNA needs to be copied. The primary enzyme

involved in synthesizing identical copy of DNA is DNA polymerase. DNA polymerase I a

bacterial polymerase was first discovered by Arthur Kornberg in 1956 and for this significant

achievement he was conferred Nobel Prize in 1959.

DNA polymerase use single strand DNA as template and catalyzes DNA dependent DNA

synthesis. This enzyme adds complementary nucleotide to 3’-OH end of newly synthesized

DNA strand. The 3’-OH group of growing DNA strand act as nucleophile and attack α-

phosphoryl group of incoming nucleotide. The reaction results in addition of new base by

formation of phosphodiester bond and release of pyrophosphate. Hydrolysis of released

pyrophosphate provides additional free energy to make this reaction irreversible with a ΔG of -7

kcal/ mole. The addition of new nucleotide is directed by template strand as the nucleotide that

can form Watson-Crick base pair with template stand is added. Figure 1 depicts the mechanism

of addition of base by DNA polymerase during DNA synthesis.

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 Figure 1. Diagrammatic representation of addition of nucleotide during DNA replication

3.2 Function of DNA polymerase

DNA polymerase performs two basic functions during DNA synthesis viz. DNA

polymerization and proofreading. Polymerization function is carried out by virtue of 5’-3’

polymerase activity. Newly incorporated nucleotide can only be added to 3’-OH group and

because of this new strand is always synthesized in 5’-3’ direction. DNA replication is a

bidirectional and semidiscontinuous process. The recently synthesized strand that moves in the

direction of replication fork opening is synthesized continuously and is called leading strand.
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 Other strand is synthesized in small stretches as the direction of DNA polymerization is

opposite to the movement of replication fork. This strand is called lagging strand and the small

stretches of DNA synthesized is called Okazaki fragments (Figure 2).

Figure 2. Bidirectional DNA polyme rization in semidiscontinuous manne r

DNA polymerase has not only the role to synthesize DNA but also to make it correctly.

Impaired synthesis of DNA can lead to altered metabolic functioning of cell and even

death. DNA polymerase has two types of exonuclease activity i.e. 3’-5’ and 5’-3’

exonuclease activity. As wrongly paired nucleotide is incorporated during DNA

synthesis, the polymerase activity is repressed and template strand moves from

polymerase active site to 3’-5’ exonuclease active site. The mismatched base is excised

and DNA synthesis resumes again. The function of DNA polymerase to remove

mismatched base pair by virtue of 3’-5’ exonuclease activity is called proofreading.

Proofreading function is performed without template strand getting dissociated from

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 DNA polymerase as polymerase and 3’-5’ exonuclease active sites are part of same

polypeptide. Proofreading machinery helps in maintaining high fidelity during DNA

replication. Generally DNA polymerase incorporates one wrong nucleotide for every

105 nucleotides added. Proofreading function improves this rate by a factor of 100 to

107 . Overall error rate of one in 10 10 nucleotide added is achieved further by post

replication repair mechanism.

5’-3’ exonuclease activity of DNA polymerase helps in nick translation process. DNA

polymerase binds to single stranded nick and removes nucleotide from 5’ end. New

nucleotides are added by polymerase activity and the nick is sealed by DNA ligase. This

machinery helps in joining lagging strands during DNA replication.

3.3 Mechanism of DNA polymerase

DNA polymerases share common structural features. Structure of DNA polymerase resembles

to right hand with three distinct domains designated as palm, finger and thumb domains

(Figure 3A). Palm domain contains the active site for polymerase activity. Two metal ions

usually Mg +2 bound to conserved aspartate catalyze nucleophilic attack of 3’ OH on phosphate

group. Metal ion A binds to 3’ OH and decreasing its affinity for H group. This result in

weakening of O-H bond and assist nucleophilic attack of 3’ OH on α-phosphate group. Metal

ion B interacts with phosphate groups and help release of pyrophosphate (Figure 3B). Palm

domain also aids in maintaining correct base pairing. It forms extensive hydrogen bonds with

Structure and Function of Biomolecules II

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DNA Polymerase
 minor groove of newly synthesized DNA, which is not base pair specific. Addition of

mismatched base pair results in reduced affinity of palm domain with primer-template junction

as well as reduced rate of catalysis. This effects release of primer-template junction from

polymerase active site to 3’-5’ exonuclease active site for removal of incorrectly paired base.

Structure and Function of Biomolecules II

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DNA Polymerase
 B

Figure 3. A. Different domains of DNA polymerase; B. Metal ions involved in catalysis

during DNA synthesis.

Finger domain interacts with incoming deoxynucleotide triphosphate (dNTP). Once correctly

paired base is added then finger domain moves to enclose the newly formed base pair. This

closed conformation increases the proximity of DNA template with active site metal ions and in

turn activates catalysis of DNA synthesis. Finger domain also interacts with template and

induces 90⁰ turn in primer template junction to expose first unpaired base for catalysis. This

avoids confusion for the site of new base addition. Thumb domain though not involved directly

in catalysis, binds with newly synthesized DNA. This interaction helps in correct positioning of

primer to active site. Thumb domain also increases the association of template with DNA

polymerase. This close association increases the processivity of DNA polymerase i.e. number
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 of base pair added each time DNA polymerase binds to DNA template. In short closed

association and coordination of palm, finger and thumb domain results in enhanced catalysis

coupled with accurate paired base addition.

3.4 Types of DNA polymerases

DNA polymerase has role to synthesize DNA with high fidelity matching the speed of cell

division. To accomplish this task cell requires different types of DNA Polymerases. Prokaryotic

and eukaryotic DNA polymerase though undertakes same function but differ in types.

3.4.1 Bacterial DNA polymerases

Five different types of polymerases designated as Pol I, II, III, IV and V has been reported in

bacteria. They have been numbered in sequence of their discovery. DNA Polymerase I (Pol I)

first discovered in Escherichia coli is 928 amino acids long single polypeptide, endowed with

three activities i.e. 5’-3’ polymerase, 3’-5’ exonuclease and 5’-3’ exonuclease. 3’-5’

exonuclease activity of Pol I supports in proofreading activity to synthesize DNA with high

fidelity. 5’-3’ polymerase and 5’-3’ exonuclease activity helps in combining and completing the

synthesis of lagging strand. 5’-3’ exonuclease activity aids in removing DNA/ RNA from nick

region particularly from DNA-RNA junction of Okazaki fragments. The gap created by this is

filled by 5’-3’ polymerase activity of Pol I. Pol I is not a highly processive enzyme and adds 20-

100 nucleotides each time it binds to template but this is ideal for filling short gaps. Pol I is

involved in nick translation and other DNA repair mechanism of cells. DNA polymerase I of E.

coli can be proteolytically cleaved into a large fragment called Klenow fragment. This

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 fragment has largely been studied as model DNA polymerase. This fragment is devoid of 5’-3’

exonuclease activity and contains polymerase and 3’-5’exonuclease catalytic sites.

DNA Polymerase II (Pol II) has function in DNA repair and cells deficient in it can propagate

usually. This is also involved in initiating replication at replication fork when it is stopped

because of DNA damage. DNA Polymerase III (Pol III) is the primary polymerase involved in

bacterial DNA replication. This is a highly processive enzyme with polymerase and 3’-5’

exonuclease catalytic site. These two activities help in DNA synthesis with high speed and

fidelity. The DNA polymerase III core enzyme contains three subunits viz. α, ε and θ.

Polymerase activity is associated with α, 3’-5’ exonuclease activity is found in ε and θ

subunit stimulates exonuclease activity. Pol III lacks 5’-3’ exonuclease activity and cannot

perform function of nick translation. The properties and functions of above three bacterial

polymerases is enlisted in Table 1.

Table 1. Properties and function of E. coli DNA polymerases

Pol I Pol II Pol III

Mass (kD) 103 90 130
Molecules/cell 400 Not defined 10-20
Turnover number 600 30 9000
Number of Subunit Core(3)
1 1
Holoenzyme (10)
Structural gene polA polB polC
Lethal mutant Yes No Yes
5' - 3' Polymerization activity Yes Yes Yes
3' - 5' Exonuclease activity Yes Yes Yes
5'- 3' Exonuclease activity Yes No No
Function RNA primer removal; DNA repair DNA Replication
DNA repair
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 DNA Polymerase IV and V are mainly involved in DNA repair. They also permit replication to

escape certain DNA damage and are called error-prone polymerases.

3.4.1.1 Assembly of DNA polymerase III holoenzyme increases processivity

DNA polymerase III being primary polymerase of bacteria need to have high processivity to do

so it forms holoenzyme an association of closely linked 10 proteins. E. coli DNA Pol III

holoenzyme is a 900 kD complex organized in four subcomplexes. Figure 4 depicts the steps

and subunits involved in holoenzyme association. The β ring a homodimer of two β subunit

form a clamp around template strand and can slide freely on it. Liaison of β ring to core complex

increases its processivity (>5000 bases) as β ring does not allow the core complex to diffuse

away from template DNA. The γ complex a cluster of seven proteins helps in placing β ring on

template strand by using ATP. Because of this function γ complex is also called clamp loader.

Once the β clamp is bound to DNA, DNA Pol III core enzyme associates with it. A τ dimer

binds to core complex and initiates linking of another core complex already bound to β

clamp. Core enzyme complex is bound to stretchy C-terminal domain of τ complex

which provides it flexibility of movement.

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 Figure 4. Steps involved in DNA Polymerase III holoenzyme association
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 DNA polymerase III needs to synthesize DNA on both leading and lagging strand in close

coordination. This task is accomplished by assembly of two DNA Pol III holoenzymes to form

replisome (Figure 5). While one core synthesizes DNA continuously on leading strand, other

core after completing one Okazaki fragment, dissociates and bind β clamp recently added on

newly exposed template. Two core complexes need to have different processivity as lagging

strand core need to dissociate each time it completes synthesis of one Okazaki fragment. The

single clamp loader of replisome is associated with core polymerase of lagging strand. Clamp

loader is also required for removing β clamp from DNA template and it has important function

of loading and unloading of β clamp after each Okazaki fragment synthesis.

Figure 5. Pictorial representation of E. coli replisome

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 3.4.2 Eukaryotic DNA polymerases

Eukaryotic genetic makeup is complex as compared to prokaryotes. The size of genome is big

and DNA replication starts at multiple origin points. In addition to this organelles DNA of

mitochondria and chloroplast also needs to be replicated. Eukaryotes require more number of

proteins for DNA replication. Eukaryotic cells need various DNA polymerases and on an

average more than 15 polymerases are present. Eukaryotic polymerases are designated by Greek

letters according to their sequence of finding. A newer classification based on sequence

homology places both eukaryotic and prokaryotic polymerases into six families i.e. A, B, C, D,

X and Y. Three main eukaryotic DNA polymerases α, δ and ε fall in B-family. Pol α/

primase is a four subunit complex consisting of two subunit each of Pol α and primase.

Pol α/ primase initiates DNA replication in eukaryotes. Primase unit synthesizes 7-10

nucleotide RNA primer and Pol α adds dNTP to it. Pol α lacks proofreading

(exonuclease) activity but this is not a problem as stretch of initially synthesized DNA

along with RNA primer is removed during processing. Pol α is a not a processive

enzyme and get replaced with polymerase δ and ε, this process is called polymerase

switching.

DNA polymerase δ is a highly processive enzyme and contains 3’-5’ exonuclease

activity. High processivity of this polymerase is due to association with eukaryotic

sliding clamp protein called proliferating cell nuclear antigen (PCNA). John Kuriyan

determined the X-ray structure of PCNA and it showed similarity with E. coli β clamp.
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 Though PCNA share common structure and function with β clamp but their sequence

identity is not similar. The eukaryotic clamp loader replication factor C (RFC) loads

PCNA to primer-template junction by hydrolysis of ATP. Pol δ is required for lagging

strand synthesis however can take part in leading strand synthesis. DNA polyme rase ε

is also a highly processive polymerase having 3’-5’ exonuclease activity. Pol ε is

needed for leading strand synthesis but it can help in lagging strand synthesis. The

properties and function of above three main eukaryotic DNA polymerase is listed in

Table 2.

Table 2. Properties and function of primary eukaryotic DNA polymerases

Pol α/ primase Pol δ Pol ε

Mass (kD) 350 250 350
Number of Subunits 4 4 4
3' - 5' Exonuclease activity No Yes Yes
Processivity Moderate High High
Association with PCNA No Yes No
Function Primer synthesis DNA replication; DNA replication;
Proofreading Proofreading

DNA polymerase γ a three subunit A- family enzyme is found in mitochondria. Pol γ is

solely involved in mitochondrial DNA replication including DNA repair and

recombination. Bulk of additional eukaryotic DNA polymerases function in DNA

repair. DNA polymerase β a 39 kD monomeric protein is high fidelity repair

polymerase. Fidelity of β matches with primary DNA polymerases. Other eukaryotic

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 DNA polymerase involved in DNA repair has much inaccuracy and are called error-

prone polyme rase.

3.4.3 Unique DNA polymerases

Some of DNA polymerases have exceptional properties and it will be worth to mention them.

One among them is Taq DNA polymerase isolated from thermophilic bacteria Thermus

aquaticus. This enzyme revolutionized the world of molecular biology by being used in

polymerase chain reaction (PCR). Taq Pol can amplify a template DNA into several copies at

high temperature. PCR the process in which this enzyme is used forms the backbone of

recombinant DNA technology. Taq pol lacks proofreading activity and its error rate is high.

Other thermotolerant DNA polymerases such as Pfu polymerase from Archaea Pyrococcus

furiosus synthesizes DNA with high fidelity and has replaced Taq Pol in PCR reaction.

One more distinctive DNA polymerase is reverse transcriptase found in retroviruses. This

catalyzes RNA dependent DNA synthesis. Using a RNA template it can synthesize

complementary DNA in 5’-3’ direction. It was first discovered by Howard Temin and David

Baltimore in 1970. Reverse transcriptase has been a useful tool of genetic engineering for

synthesizing complementary DNA (cDNA) from mRNA (Figure 6). As mRNA does not

contain introns so, cDNA can be used for expressing eukaryotic proteins in E. coli which lacks

splicing machinery.

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 Figure 6. Illustration of cDNA synthesis from eukaryotic mRNA by use of reverse
transcriptase

Telomerase is a DNA polymerase used exclusively to synthesize DNA at ends of linear

eukaryotic chromosomes called telomere. Lagging strand synthesis in eukaryotes pose a

problem as 5’ end cannot be synthesized and each cycle will lead to shortening of chromosome

equivalent to the length of RNA primer. Shortening of end of chromosome after each cycle can

cause cell death. This problem is overcome by ribonucleoprotein telomerase. Telomeres contain

a >1000 tandem repeats of G-rich sequence i.e. TTGGGG in Tetrahymena and TTAGGG in

human. RNA component of telomerase contain sequence similar to telomeres and act as

template for addition of dNTP at 3’ end of DNA. Figure 7 illustrates telomerase action to

synthesize ends of linear eukaryotic chromosome.

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 Figure 7. Graphic representation of telomere DNA synthesis by Tetrahymena
telomerase

By multiple round of replication telomerase synthesizes the end of chromosome leaving a G-

rich overhang. Enhanced telomerase activity can cause uncontrolled cell growth and can result

to cancer. Function of telomerase is related to reverse transcriptase and its highly conserved

catalytic subunit TERT is homologous to reverse transcriptase.

4. Summary

In this lecture we learnt about:

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  Functions of DNA polymerase i.e. Polymerase, proofreading and nick translation

 Mechanism of catalysis by DNA polymerase; role of palm, finger and thumb domain; function of active

site metal ions.

 Different types of Bacterial polymerases

 DNA Polymerase III holoenzyme

 Eukaryotic DNA polymerases

 Special DNA Polymerase: Taq polymerase; Reverse transcriptase; Telomerase

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